Method for inhibiting platelet interaction with biomaterial surfaces

ABSTRACT

A method for passivating a biomaterial surface includes modifying proteinaceous material disposed at the biomaterial surface. The passivation may be effectuated by exposing the biomaterial surface to therapeutic electrical energy in the presence of blood or plasma.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Continuation of and claims priority to U.S. patentapplication Ser. No. 12/057,729, filed Mar. 28, 2008, now allowed, whichclaims priority from U.S. provisional application Ser. No. 60/908,576,filed on Mar. 28, 2007, the disclosures of which are incorporated hereinby reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to providing biomaterial surfaces withthromboresistivity generally, and more particularly to materials andmethods for passivating a biomaterial surface so as to inhibit bloodplatelet interaction therewith.

BACKGROUND OF THE INVENTION

Since the year 2000 alone, more than 1,000,000 vascular prostheticdevices have been implanted worldwide. From stents to artificial heartvalves and ventricular assist devices, a wide range of devices are beingused to treat patients often expected to live for many years after theprocedures. Since biomaterials promote surface-induced thromboticphenomena to some extent, an ever-increasing pool of patients reliantupon indefinite anticoagulant therapy has been created. This isunfortunate, as the use of drugs like heparin, warfarin and clopidogrelcarries a serious risk of side effects like bleeding, bruising andserious internal hemorrhage.

Blood contacting biomaterial surfaces in particular, have been shown toadsorb a layer of proteins from blood and to attract platelets. Build-upof blood components on the surface of implanted devices may reduce theireffectiveness, and in many cases will lead to serious adversecomplications or operational failure. Thrombogenesis presents a majorproblem associated with the clinical use of all kinds of prosthetics,and the prevention of unwanted clotting without the side effectsincurred through the use of blood thinning drugs would be a majoradvancement in the field of biomaterials.

One method for securing biomaterials against unwanted thrombosis is tomodify the biomaterial surface itself. For example, anti-thrombogenicmaterials have been covalently bonded onto the blood-contactingbiomaterial surfaces. Additionally, the biomaterial has been treated togive its surface a fixed charge which can affect the biocompatibility ofthe material. In other cases, the surface has been polished to anextremely high degree. None of these techniques, however, have beencompletely effective in deterring platelet adhesion to the biomaterialsurface.

It has been theorized that promoting adhesion of albumin to thedetriment of fibrinogen at the blood-contacting surface could beeffective in altering the thrombogenicity of various materials. In fact,Grunkemeier et al. Biomaterials, November 2000 pp. 2243-2252, and Tsaiet al., Journal of Biomedical Materials Research Dec. 15, 2003, pp.1255-68, found that the amount of adsorbed fibrinogen was the chiefdeterminant of the degree of platelet adhesion, although platelets weremost attracted to a surface when a combination of proteins was residingon the surface, including von Willebrand factor. No preadsorption ofparticular blood proteins has yet been shown to prevent clottingentirely. It is very difficult to prevent fibrinogen from adhering tothe biomaterial surface, and only a small amount of adhered fibrinogenis necessary to start a chain reaction leading to thrombosis.

Some materials coated with anticoagulant agents such as heparin have hadlimited success in preventing thrombosis. However, heparin coatings willeventually dissolve over time. Drawbacks to agent-eluting surfaces havealso been realized. A study by Pfisterer et al., Journal of AmericanCollege of Cardiologists, Dec. 19, 2006 pp. 2592-5 regarding the BaselStent Kosten Effektivitats Trial, Late Thrombotic Events, suggested thatbetween 7 and 18 months after implantation, the rates of nonfatalmyocardial infarction, death from cardiac causes, and angiographicallydocumented stent thrombosis were higher with drug-eluting stents thanwith bare metal stents.

Overall, there have been no recognized clinical advancements that couldwarrant replacing traditional anticoagulation therapy. At this time,only consistent maintenance of a regiment of blood thinning agents isclinically proven to prevent the dangerous thrombotic events associatedwith implants.

It is a primary object of the present invention to inhibit and/orprevent thrombogenesis and blood platelet adhesion on a biomaterialsurface.

It is another object of the present invention to inhibit and/or preventblood platelet adhesion and thrombogenesis on an electricallyconductive, blood-contacting surface of an implantable device.

It is a further object of the present invention to provide ananti-thrombogenic characteristic to biomaterial surfaces by providingcertain blood proteins thereat.

It is a further object of the present invention to provide ananti-thrombogenic characteristic to biomaterial surfaces, by providingconformationally-modified blood proteins thereat.

It is a still further object of the present invention to provide amethod to pre-treat biomaterials such as pyrolytic carbon, titanium,nitinol, and stainless steel using therapeutic electrical energy so asto prevent blood platelet adhesion to the pre-treated biomaterials.

SUMMARY OF THE INVENTION

By means of the present invention, biomaterial surfaces may be providedwith a thromboresistant characteristic, such that blood-contactingsurfaces of a biomaterial inhibits blood platelet interaction andadhesion therewith. Such passivation of the biomaterial surface iseffectuated through a passivating procedure, which may involveapplication of therapeutic electrical energy and/or deposition ofcertain proteinacecous material thereat. Biomaterial surface passivationmay be accomplished in vivo, ex-vivo, or in vitro, and may be done priorto, or subsequent to implantation of a biomaterial in a patient.

In one embodiment, a method for passivating a biomaterial surfaceinvolves exposing the biomaterial surface to therapeutic electricalenergy in the presence of blood or plasma.

Another method for passivating a biomaterial surface includes modifyingproteinaceous material disposed thereat.

The biomaterial surface may also be passivated by exposing a quantity ofblood or plasma to therapeutic electrical energy and subsequentlydepositing proteinaceous material from such quantity of blood or plasmaon the biomaterial surface.

A biomaterial surface effective in inhibiting blood platelet adhesionthereto includes conformationally modified fibrinogen.

A still further method for passivating a biomaterial surface includesmodulating a preferential adsorption of blood proteins to thebiomaterial surface.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of a testing apparatus used in the testingof the materials and methods of the present invention;

FIG. 2 is a partial cut-away view of a portion of the testing apparatusillustrated in FIG. 1;

FIG. 3 is a process flow diagram of the testing procedure for testingthe methods and materials of the present invention;

FIG. 4 is a SEM image comparison between a first control test articleexposed to whole human blood and a second control test article notexposed to blood;

FIG. 5 is a SEM image comparison between a third control test articleexposed to whole human blood in an unstimulated environment, with afourth test article exposed to whole human blood in a stimulatedenvironment;

FIG. 6 is a series of gel electrophoresis images comparing surfaces oftest articles in a stimulated environment with test articles in anunstimulated environment;

FIG. 7 is a radioactive count chart illustrating platelet concentrationsat test articles exposed to different test environments.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The objects and advantages enumerated above together with other objects,features, and advances represented by the present invention will now bepresented in terms of detailed embodiments. Other embodiments andaspects of the invention are recognized as being within the grasp ofthose having ordinary skill in the art.

The present invention is drawn to techniques and materials which havebeen found to be useful in inhibiting platelet interaction withbiomaterial surfaces. Such interaction may include, for example,adhesion, aggregation, thrombosis, clotting, and/or coagulation of bloodplatelets at a biomaterial surface exposed to such platelets. For thepurposes of this application, the terms “passivate”, “passivated”, or“passivating” shall refer to a surface that exhibits anti-thrombogenicproperties so as to inhibit thrombosis thereat. In some instances, suchterm may further connote improving the biocompatibility of the surface,such as through thromboresistant properties. For the purposes of thisapplication, “thromboresistant” and “anti-thrombogenic” may be usedinterchangeably.

A variety of biomaterials may be passivated through the presentinvention. Most commonly, however, biomaterials include those materialsthought to be useful in the fabrication of medical articles, such asimplantable medical articles. However, the techniques and materials ofthe present invention may indeed facilitate the use of “biomaterials”which, in the absence of the techniques and materials of the presentinvention, would not typically be considered in medical applications,such as in implantable medical articles. Accordingly, as used herein,“biomaterials” is intended to include any native, natural, and/orartificial material used in a biological application, such as in thecontacting of blood, plasma, or other biological fluids. Examplebiomaterials may include metal such as stainless steel, nitinol, andtitanium, plastics such as polyolefins, polyesters, polystyrenes,polyurethanes, polyamides, polyacrylonitriles, polymethacrylates,silicones, and silicone rubbers, as well as other materials such aspyrolytic carbon and ceramics. In some embodiments of the invention,electrically conductive materials, such as those having an electricalresistivity of less than about 5 ohms may be utilized, though suchresistivity threshold may be overcome by using vascular stents, grafts,heart valves, heart diaphragms, catheters, implantable pacemakers,defibrillators, and related leads, sutures, needles, tubing, dialysismembranes, filters, and the like.

The Applicant has focused studies on the thromboresistant propertiesexhibited by proteinaceous materials contained in or derived from blood.Example such proteinaceous materials that have been addressed in thiseffort include albumin and fibrinogen. One thromboresistant factor thathas been developed in the present studies is a conformationally alteredfibrinogen. In its standard form, fibrinogen includes three distinct“conformations”, each having slightly different molecular weights. Thesemay be referred to as alpha, beta, or gamma fibrinogen. Such distinctconformations can be viewed through gel electrophoresis, wherein threedistinct bands are prevalent at between about 50 kD. Subsequent toexposure to the electrical energy as described herein, the relativeconcentrations of the fibrinogen conformations at the biomaterialsurface are modified. In one embodiment, at least one of the threefibrinogen conformations exposed to the therapeutic electrical energy isfound at significantly higher concentrations at a surface exposed to theelectrical energy than the concentration of such fibrinogen conformationat a surface not exposed to the therapeutic electrical energy. Inaddition, at least one fibrinogen conformation concentration issignificantly decreased upon electrical stimulation. It is hypothesizedthat the binding factor of fibrinogen to blood platelets is modified oreliminated through the fibrinogen has little or no adhering interactionwith blood platelets, thereby effectuating a thromboresistantcharacteristic.

Another thromboresistant factor of the present invention is thepreferential promotion of albumin adhesion to a biomaterial surface.Biomaterials having a relatively high surface concentration of albuminhave been shown to inhibit fibrin cascade and platelet attachment,potentially through disruption of electric charge-related plateletinteractions. Typically, however, fibrinogen is often the dominantprotein adsorbed from protein mixtures such as blood, blood serum, orplasma. Because fibrinogen, in its standard form, is known to promoteplatelet adhesion at a surface, preferential albumin adsorption acts toinhibit platelet adhesion both through the thromboresistant propertiesof albumin and through the reduction of fibrinogen presence at thesurface.

One technique for effectuating one or more of the thromboresistantfactors described above at a biomaterial surface involves theapplication of electrical energy to proteinaceous material found inblood and/or the application of electrical energy to a biomaterialsurface while in the presence of such proteinaceous material. Theapplication of electrical energy, such as a magnitude of electricalenergy deemed therapeutic, to blood or plasma has surprisingly beenfound to cause thromboresistance in biomaterial surfaces contacted withthe treated blood or plasma. It is theorized that the thromboresistancegenerated at the biomaterial surface is derived from one or both of thepresence of conformationally altered fibrinogen and thedisproportionately high concentration ratio of albumin to standardfibrinogen at such biomaterial surface. Moreover, it is theorized thatthe existence of such thromboresistant factors at the biomaterialsurface is created through the application of therapeutic electricalenergy to proteinaceous material contained in blood, blood serum, orplasma, wherein such proteinaceous material includes albumin and/orfibrinogen. Through experimentation, Applicant has determined thatapplication of electrical energy, in the therapeutic magnitudesdescribed herein, establishes an environment for the creation of athromboresistant, passivated biomaterial surface characteristic.Applicant contemplates, however, that alternative methods may beemployed to establish the thromboresistant factors described herein, andto provide a biomaterial surface with one or more of such factors.

In one embodiment, a biomaterial surface may be passivated by exposingsuch biomaterial surface to therapeutic electrical energy in thepresence of blood or plasma. In another embodiment, a biomaterialsurface may be passivated by exposing such biomaterial surface to bloodor plasma which has been treated with therapeutic electrical energy. Ina further embodiment, a biomaterial surface may be passivated byadsorbing at such surface blood proteins treated with therapeuticelectrical energy. In another embodiment, a thromboresistant biomaterialsurface may be achieved through the provision of a conformationallyaltered fibrinogen thereat. Other embodiments in addition to thosedescribed above are also contemplated as being within the scope of thepresent invention.

Investigations have been conducted into the prevention of biomaterialsurface/platelet interaction with the application of electric current tovarious materials. Initial studies have focused on the reaction ofpyrolytic carbon, stainless steel, nitinol, and titanium. Currentlymarketed cardiac and vascular stents are primarily made of stainlesssteel (also carbon coated), and nitinol. In initial experiments,pyrolytic carbon was chosen one to previous experience with thismaterial. The type and magnitude of electrical energy (frequency andcurrent) needed to provide thromboresistance on the surface of carbonhave been investigated using an in-vitro blood perfusion system, asdescribed below. Assessment of the reactions has been accomplishedthrough scanning electron microscopy (SEM), electrophoresis, Indium(radioactive) platelet labeling, protein, assay assessment, andFluorochrome-labeled antibody staining.

Test System

A blood perfusion system (BPS) was developed for the evaluation of thebiomaterial surfaces and its reaction to blood, and was designed to holdany one of the biomaterials of interest. A schematic diagram of bloodperfusion system 10 is illustrated in FIG. 1, and includes a samplereservoir 12, a test chamber 14, and a fluid pump 16 for pumping samplefluid throughout system 10. An electrical power supply 18 iselectrically coupled to test chamber 14 through electrical leads 20, 22,and may controllably apply electrical energy to test chamber 14. System10 further includes fluid conduit sections 30, 32, 34 for transportingthe sample fluid throughout system 10.

Sample reservoir 12 of system 10 may be any type of reservoir for thefluids utilized in the test procedure. By way example, such fluids mayinclude whole blood, platelet-rich plasma, or platelet-poor plasma. Insome cases, a suspension such as sodium citrate or sodium heparin may beadded to the fluid to inhibit spontaneous clotting. Sample reservoir 12in the test, apparatus was a 0.5 liter glass bottle.

Both, a pulsatile pump and a roller pump were utilized as pump 16 ofsystem 10. The roller pump, which was a Model 232 pump manufactured byWatson Marlow was utilized in continuous flow regimes at a flow rate of600 ml per minute. A MOX106 pulsatile pump manufactured by WatersInstruments was calibrated to mimic a beating human heart, wherein apump surge rate of 70-80 surges per minute was set with an output volumeof 50 ml per surge. Pump 16 pumped the sample fluid throughout conduitsections 30, 32, 34, which comprise silicon rubber tubing. Inparticular, pump 16 pumps the sample fluid from sample reservoir 12 totest chamber 14, and then back to sample reservoir 12.

Test chamber 14 is illustrated in greater detail in FIG. 2, wherein testchamber 14 includes a polycarbonate housing 42, a top lid 44 and abottom lid 46. The top and bottom polycarbonate lids 44, 46 aresealingly engageable with housing 42 via O-rings 48. The sample fluid issupplied to test chamber 14 at fluid inlet 50, and is removed from testchamber 14 at fluid outlet 52. Valves 54 were positioned at fluid inletoutlet 50, 52 for additional control of fluid flow through test chamber14. The biomaterial surface analyzed in test chamber 14 was a prostheticbi-leaflet heart valve 56 fabricated from pyrolytic carbon, and furtherprovided with a fabric suture cuff 58 in conventional fashion. Theprosthetic heart valve was a 25 mm ATS Open Pivot™ aortic valve having aleaflet surface area of about 12.4 cm².

To suspend the valve prosthesis within test chamber 14, a titanium pinretainer 60 with electrically insulative plastic covers 62 were retainedat apertures 43 of housing 42, and pierced the fabric suture cuff 58 ofvalve prosthesis 56. At least one pin retainer 60 was placed intocontact with the pyrolytic carbon body of valve prosthesis 56 so as tomake electrical contact to at least the valve body of valve prosthesis56. In addition, such as least one pin retainer includes an exposedextension portion 64 to which electrical connection may be made. Thispin retainer 60 thus forms an electrode for establishing directelectrical contact with valve prosthesis 56. A further electrode 70 isprovided through an aperture 45 in top lid 44, with titanium electrode70 extending into the chamber defined by housing 42 and into contactwith the valves leaflets of valve prosthesis 56. In this manner, directelectrical contact to valve prothesis 56 could be established byconnecting an electrical lead to connection end 72 of electrode 70. Aset screw 76 was utilized in order to adjust the vertical position ofelectrode 70 within test chamber 14, and particularly into and out fromelectrical contact with valve prosthesis 56.

A still further electrode 80 was provided in test chamber 14, whereintitanium electrode 80 is exposed to the sample fluid 13, but is spacedfrom valve prosthesis 56. Electrical connection to electrode 80 could bemade at connection end 82 thereof. As illustrated in FIG. 2, the levelof sample fluid 13 was above valve prosthesis 56, such that valveprosthesis 56 was submerged in the sample fluid 13 during the testprocedure.

Power supply 18 was a combination of a Tektronix™ AFG310 arbitrarywaveform generator which is capable of producing multiple electricalwaveforms (sin, triangular, square, and pulsatile) and a customprecision voltage to current converter capable of delivering variouscurrents level. Electrical leads from power supple 18 were connected torespective ones of the electrodes 60, 70, 80 during the test procedure.In some cases, positive polarity was coupled to both electrodes 60 and70 while negative polarity was coupled to electrode 80. In other cases,positive polarity was coupled only to electrode 70 while negativepolarity was coupled to electrode 80. Electrical connection wasestablished at the terminus of the electrical leads through conventionalelectrical clips.

Test Procedure

The evaluation procedure was as follows:

A test article was inspected and cleaned with alcohol, and then placedin test chamber 14 as described above. A 250 ml reservoir of saline wasplaced in a water bath at 37° C. Once the saline reached equilibriumtemperature, the open ends of conduit sections 30, 34 were placed in thereservoir. Pump 16 was activated and adjusted to a flow rate of 600 mlper minute to pump the saline through system 10 for ten minutes to rinsethe system and to test for potential leakage.

In “direct connection” tests, positive polarity electrical connectionsare made to electrodes 60, 70, and a negative polarity electricalconnection is made to electrode 80. Moreover, in “direct connection”tests, electrode 70 is positioned so as to make direct contact with atleast a portion of the test biomaterial article. In the case of valveprosthesis 56, electrode 70 may be placed in direct contact with thepyrolytic carbon leaflets when conducting a “direct connection” test.

In “electrical field” tests, positive polarity electrical connection ismade to electrode 70 and negative polarity electrical connection is madeto electrode 80. Electrode 70 is vertically positioned within testchamber 14 so as to be out of contact with the test article during theapplication of electrical energy.

Power supply 18 was calibrated to provide a signal having positive going(2.25 V/2.25 mA DC offset, a 4.5 V peak pulse which correlates to a 4.5mA current. The current is derived by making a differential measurementof the signal across a precision 1 kΩ resistor. A duty cycle of 41.6%was assigned (25 ms ON (+4.5 V) and 60 ms OFF (0 V)).

Pump 16 is then turned off and system 10 drained of the saline. A 250 mlreservoir of sample fluid (human whole blood, animal whole blood, bloodserum, platelet rich plasma, platelet poor plasma, etc.) replaces thesaline reservoir in the water bath set to 37° C. Pump 16 is againactivated to expose system 10 to the sample fluid. Upon completion ofthe test period, the sample fluid is drained from system 10 and system10 is then immediately flushed with saline through the process describedabove.

The test article is then removed from test chamber 14, rinsed in saline,and placed in a solution of gluteraldehyde to arrest further cell actionand interaction. The test article is then dehydrated with ethanol toenable assessment of the article surface within the scanning electronmicroscope vacuum chamber.

EXAMPLES

The invention is further and more specifically illustrated by thefollowing examples and tests.

Example 1

A control experiment was conducted using whole human blood donatedwithin three hours of testing. The whole human blood was pumped throughsystem 10 in the absence of applied electrical energy, and was contactedwith a pyrolytic carbon heart valve prostheses at test chamber 14. Thistest was continued for 30 minutes.

FIG. 4 illustrates two scanning electron microscope (SEM) slides takenat 1000× magnification. A photograph of a clean, untested pyrolyticcarbon valve prosthesis leaflet is shown of the left, and a pyrolyticcarbon valve prosthesis leaflet taken from the test article followingthe control test is shown on the right. It is clear from this controlsample that blood platelets are adhered and spread across the surface ofthe pyrolytic carbon under typical blood exposure conditions, such asthose found in vivo. The conditions of the control experimentsubstantially replicate conditions experienced in vivo for implantablemedical articles.

Example 2

A sample of whole human blood was separated into two aliquots, with afirst aliquot being tested through the “direct connection” proceduredescribed above for 45 minutes. The second aliquot of whole human bloodwas cycled through system 10 and contacted with a pyrolytic carbon testarticle in the absence of electrical energy application as a control for45 minutes. The SEM slides of FIG. 5 demonstrate an image of the controltest article surface on the right, and an image of the test articlesurface used in the “direction connection” test on the left.

It is clear from visual comparison of the SEM slide that the testarticle surface exposed to the electrical energy is substantially clearof adhered platelets, while the control test article exhibitssignificant platelet confluency at its surface. A graphical pixilationanalysis was performed to derive a quantitation of blood platelet cellpresence at the respective test article surfaces. The graphicalpixilation analysis was performed by colored pixel count of the SEMimages, wherein individual pixel colors other than black were consideredadhered platelet cells. The graphical pixilation count analysis of testarticle surface exposed to electrical energy revealed about 2.5%platelet adhesion, while the control test article surface exhibitedabout 59.1% platelet cell confluency.

Example 3

A sample of human platelet rich plasma (PRP) was separated into threealiquots with a first aliquot being tested through the “directconnection” procedure described above in a “stagnant” flow regime,wherein the test articles are exposed to a stagnant volume of test fluidfor the test period. A first pyrolytic carbon article was exposed to thefirst aliquot of PRP in the presence of the electrical energyapplication described above for 15 minutes. The first pyrolytic carbontest article was then exposed to whole human blood from the PRP donor ina pulsed flow regime for 45 minutes in the absence of applied electricalenergy.

A second pyrolytic carbon test article was tested similarly to the firstpyrolytic carbon test article, except that the second test article wasexposed to PRP in the presence of applied electrical energy for 30minutes prior to exposure to whole human blood from the PRP donor for 45minutes in the absence of applied electrical energy.

Control pyrolytic carbon test article were exposed to PRP in thestagnant chamber for 15 and 30 minutes, respectively, without appliedelectrical energy, and then exposed to whole human blood from the RPdonor in a pulsed flow regime for 45 minutes in the absence of appliedelectrical energy.

The test article surfaces were assessed with SEM, and the first andsecond test articles exhibited significantly less adhered platelets thanthe amount of adhered platelets observed on the control test article.

Example 4

A sample of human platelet poor plasma (PPP) was separated into threealiquots for testing in connection with three test articles. A firstpyrolytic carbon test article was exposed to the first aliquot of PPP inthe presence of direct connection electrical energy for 15 minutes. Thefirst pyrolytic carbon test article was then exposed to whole humanblood from the PPP donor in a pulsed flow regime for 45 minutes in theabsence of applied electrical energy.

A second pyrolytic carbon test article was exposed to PPP in thepresence of applied electrical energy for 30 minutes prior to exposureto whole human blood from the PPP donor for 45 minutes in the absence ofapplied electrical energy.

Control pyrolytic carbon test articles were exposed to PPP in thestagnant chamber for 15 and 30 minutes, respectively, without appliedelectrical energy, and then exposed to whole human blood from the PPPdonor in a pulsed flow regime for 45 minutes in the absence of appliedelectrical energy.

The test article surfaces were assessed with SEM, and the first andsecond test articles exhibited significantly less adhered platelets thanthe amount if adhered platelets observed on the control test article.

Example 5

Bovine blood platelets labeled with indium-111 were used as the samplefluid in a test to determine the ability of passivated test articlesurfaces to remain effective in the prevention of platelet adhesion overtime without continued electrical stimulation. In this “pre-treatment”exercise, four pyrolytic carbon test articles were exposed to the bovineblood for 60 minutes. One of such test articles was electricallystimulated for the entire 60 minute test period. Two test articles werestimulated for 30 minutes during the bovine blood exposure, and thendisconnected from the electrical energy for the remaining 30 minutes ofthe test period. One test article was unstimulated throughout the entire60 minute test period. The electrical stimulation was conducted at theparameters described above.

The chart of FIG. 7 illustrates radioactive counts for each of the threetest groups described above, wherein the radioactive counts areindicative of platelet concentration at the respective test articlesurface. As demonstrated therein, it appears that pre-treatment of thetest article with stimulation in the presence of blood is also effectivein inhibiting platelet adhesion even in the absence of continuedelectrical stimulation. Specifically, the “Group 2” test articlesurfaces, which were electrically stimulated only for the first 30minutes of the 60 minute test period, exhibited post-test plateletconcentrations similar to the post-test platelet concentrations at thetest article surfaces of “Group 1”, which received electricalstimulation throughout the 60 minute test period. By contrast, the“Group 3” test article surfaces, which were exposed to blood in theabsence of electrical stimulation, exhibited post-test plateletconcentrations several fold higher than the platelet concentrationsexhibited by either of the stimulated group test articles.

Analysis

A gel electrophoresis analysis was performed on the test articlesurfaces regarding the blood proteins present thereat. Gelelectrophoresis was performed through the use of the microplatesprocedure of a BCA™ protein assay kit available from Pierce, a divisionof Thermo Fisher Scientific, Inc., of Rockford, Ill. Proteins taken from6 test articles, 3 of which were tested in a “stimulated” environment,and the remaining 3 were tested in an “unstimulated” environment bybeing exposed to blood in the absence of applied electrical energy.

FIG. 6 illustrates results for “stimulated” samples (those tested with,exposure to therapeutic electrical energy) versus unstimulated samples(control). The dark bands between 65 and 75 kD indicate the presence ofalbumin. It appears that the presence of albumin is enhanced by 5-10× inthe stimulated group, based on the results of the protein assay. It iswell understood that albumin stabilizes charges on materials therebypreventing electric charge-related platelet interactions. The presenceof albumin at the surface therefore plays a role in inhibiting plateletinteraction/adhesion with the test article surface. It is a surprisingresult of the above-described tests, however, that application of theutilized levels of electrical energy modulates the preferentialadsorption of at least albumin to the test article surface, in thatalbumin adsorption appears to be significantly preferentially promoted.Such preferential promotion of albumin adsorption is demonstrated by theenhanced albumin presence in the gel electrophoresis slides illustratedin FIG. 6, as well as in the protein assay analyses. It is theorizedthat the preferential promotion of at least albumin adsorption on thestimulated article surface is caused by the electrical charge providedat the surface through the applied electrical energy.

FIG. 6 further illustrates a conformational alteration of fibrinogen inthe stimulated group, as compared to the fibrinogen found on theunstimulated test article surfaces. As described above, the three bandsaround 50 kD represent alpha, beta, and gamma fibrinogen. Theunstimulated article surfaces exhibit all three fibrinogen conformationswith approximately similar intensity response through gelelectrophoresis. The stimulated test article surfaces, however,exhibited a significantly higher concentration of beta fibrinogen, and alower concentration of at least alpha fibrinogen and possibly a lowerconcentration of gamma fibrinogen as well. Of the three fibrinogenconformations, alpha fibrinogen is the lowest molecular weight, andgamma fibrinogen is the highest molecular weight. The concentrationchanges illustrated by the gel electrophoresis intensity changes in FIG.6 likely reflects a conformational alteration of the fibrinogen that isrelated to or induced by electrical stimulation.

The protein assay described above further confirms a substantialincrease in fibrinogen concentration at the stimulated test articlesurfaces, as compared to the fibrinogen concentrations found on theunstimulated test article surfaces. The fibrinogen detected at thesurfaces at the stimulated group, however, was conformationally alteredas described above. It was determined that the fibrinogen concentrationof the stimulated group was 5-10× greater than the fibrinogenconcentration of the unstimulated group, thus evidencing a preferentialpromotion of at least conformationally altered fibrinogen adsorption onthe stimulated article surface.

It is theorized that the alteration of fibrinogen receptors caused bythe exposure to the therapeutic electrical energy inhibits the bindingof further fibrinogen to the surface. The alpha chain of fibrinogencontains the RGDS sequence necessary for the platelet interactions. Thegamma chain of fibrinogen holds the dodecapeptide sequence (includingthe RGDS) that can be used for platelet aggregation. Reducing ofeliminating the presence of alpha and/or gamma fibrinogen, as seen inthe gel electrophoersis images of the stimulated group, may thereforecorrespondingly inhibit platelet adhesion and aggregation.

Commentary

In view of the above examples and analysis, Applicant has determinedthat, in one embodiment, therapeutic electrical energy applied to abiomaterial surface, while such surface is exposed to a bloodprotein-containing fluid such as whole blood or plasma, can passify suchbiomaterial surface, at least against thrombosis. These studies havefurther shown that biomaterial surface passivation may be accomplishedin a “pre-treatment” arrangement, wherein the biomaterial surfaceundergoes a passivating procedure and is subsequently placed into ablood platelet-contacting environment. The passivated biomaterialsurface exhibits ongoing thromboresistant properties even in the absenceof continuing surface passivation. In effect, therefore, a biomaterialsurface may be passivated in advance of implantation, with thepassivated biomaterial surface remaining effective, at least in the caseof thromboresistance, for a significant length of time subsequent toimplantation. Accordingly, the methods and materials of the resentinvention may be utilized, for example to prevent thrombosis formationin devices such as vascular stents by pre-treating blood-contactingsurfaces thereof in the presence of a relatively small amount of therespective patient's blood or plasma prior to device implantation intothe patient.

The electrical stimulation described above with reference to theexamples, is merely representative of various electrical energymagnitudes that may be useful in passivating biomaterial surfaces. Forexample, the applied electrical current in the above examples ofelectropositive 4.5 mA directed to a pyrolytic carbon material having asurface area of about 12.4 cm² provides a current density of about 0.35mA/cm². As described in our co-pending patent application Ser. No.11/402,463 entitled “System for Conditioning Surfaces in Vivo”, thecontent of which being incorporated herein by reference, anelectropositive current density of between about 0.001 and about 1.0mA/cm² may also be useful in the present application. Applicant believesthat a current density of at least about 0.1 mA/cm² may be mostbeneficial for the purposes described herein, depending upon theelectrical conductivities of the biomaterials at issue. An upperthreshold on the electropositive current density provided at thebiomaterial surface for therapeutic conditioning thereof in the presentapplication may be limited only by the current density threshold abovewhich undesired and/or permanent damage to such biomaterial orinterfacing material may occur.

For the purposes of this application, therefore, the term “therapeuticelectrical energy” shall mean electrical energy that is effective ingenerating an electropositive current density at the subject biomaterialsurface at a magnitude sufficient to passivate the biomaterial surfacethrough exposure of the biomaterial surface to the electrical energy inthe presence of blood or plasma. In one embodiment, such therapeuticelectrical energy results in an electropositive current density at thebiomaterial surface being treated of at least about 0.1 mA/cm².

As also described above, a further aspect of the present invention isthe surprising finding that the deposition of certain blood proteinsand/or blood protein concentrations at a biomaterial surface iseffective in passivating such biomaterial surface. One passivatingmaterial of the present invention is a conformationally alteredfibrinogen, and specifically a fibrinogen with a relatively highconcentration of beta fibrinogen and/or relatively low concentration ofalpha fibrinogen and/or gamma fibrinogen. Applicant has determined thatthe presence of such modified fibrinogen at the biomaterial surface at aconcentration of at least about 5-10× the concentration of unmodifiedfibrinogen at an unstimulated surface exposed to blood or plasma for atleast about 15 minutes may be effective in passivating the biomaterialsurface. As such, Applicant envisions a variety of techniques fordepositing a passivating agent, such as conformationally modifiedfibrinogen, at the biomaterial surface for passivating such biomaterialsurface. For example, such a passivating agent may be isolated and usedas needed, such as by depositing the passivating agent at a biomaterialsurface prior to biomaterial implantation into a patient. Such treatmentof the blood-contacting surface of the biomaterial may be performedimmediately prior to implantation or significantly prior toimplantation, with the passivating agent retaining its passivatingproperties for a significant period of time subsequent to implantation.

An overall impact, therefore, of the present invention is the preventionof platelet adhesion and thrombogenesis on biomaterial surfaces,including artificial implants, transplants, and native tissue, throughthe provision of certain modified and/or unmodified blood proteins atsuch surfaces. In one embodiment, such blood proteins may be provided atthe biomaterial surfaces through the application of therapeuticelectrical energy to the surface while the surface is in the presence ofblood or plasma. Other techniques, however, for the provision ofeffective passivating agents on target surfaces are envisioned in thepresent invention.

The invention has been described herein in considerable detail in orderto comply with the patent statutes, and to provide those skilled in theart with the information needed to apply the novel principles and toconstruct and use embodiments of the invention as required. However, itis to be understood that various modifications to the invention can beaccomplished without departing from the scope of the invention itself.

What is claimed is:
 1. A medical device comprising: a biomaterialsurface; and a layer of proteinaceous material adhered to thebiomaterial surface, the proteinaceous material including at least oneof conformationally altered fibrinogen and albumin, the proteinaceousmaterial configured to exhibit at least one anti-thrombogenic propertyat the biomaterial surface following placement of the biomaterialsurface within a human body, wherein the biomaterial surface is exposedto an electrical energy in the presence of a proteinaceous fluid tocause proteinaceous material to adhere to the biomaterial surface priorto placement of the biomaterial surface within the human body.
 2. Themedical device of claim 1, wherein the biomaterial surface has anelectrical resistivity of less than about 5 ohms.
 3. The medical deviceof claim 1, wherein the biomaterial surface is selected from the groupconsisting of pyrolytic carbon, titanium, nitinol, stainless steel,platinum, and iridium.
 4. The medical device of claim 1, wherein theelectrical energy exposure is effected through direct electrical contactbetween an electrical energy source and the biomaterial surface.
 5. Themedical device of claim 1, wherein the proteinaceous material comprisesconformationally altered fibrinogen.
 6. The medical device of claim 5,wherein the conformationally altered fibrinogen comprises a higherconcentration of beta fibrinogen as compared to a concentration of alphafibrinogen and a concentration of gamma fibrinogen.
 7. The medicaldevice of claim 1, wherein the proteinaceous material comprises albumin.8. The medical device of claim 1, wherein the proteinaceous materialcomprises albumin and fibrinogen.
 9. The medical device of claim 1,wherein the proteinaceous fluid is selected from the group consisting ofblood, plasma, blood serum, a fluid comprising a blood protein, a fluidcomprising albumin, and a fluid comprising fibrinogen.
 10. The medicaldevice of claim 1, wherein the medical device is selected from the groupconsisting of a heart valve, a vascular stent, a graft, a catheter, anda device comprising a stent.
 11. The medical device of claim 1, whereinthe at least one anti-thrombogenic property is selected form the groupconsisting of inhibiting platelet adhesion, inhibiting plateletaggregation, inhibiting thrombosis, inhibiting clotting, and inhibitingcoagulation.
 12. A medical device comprising: a biomaterial surface; anda layer of proteinaceous material adhered to the biomaterial surface,the proteinaceous material including at least one of conformationallyaltered fibrinogen and albumin, the proteinaceous material configured toexhibit at least one anti-thrombogenic property at the biomaterialsurface following placement of the biomaterial surface within a humanbody, wherein the biomaterial surface is exposed to a treatedproteinaceous fluid prior to placement of the biomaterial surface withinthe human body, wherein treatment of the proteinaceous fluid comprisesexposing the proteinaceous fluid to an electrical energy.
 13. Themedical device of claim 12, wherein the biomaterial surface is selectedfrom the group consisting of metals, plastics, ceramics, and pyrolyticcarbon.
 14. The medical device of claim 12, wherein the proteinaceousmaterial comprises conformationally altered fibrinogen.
 15. The medicaldevice of claim 14, wherein the conformationally altered fibrinogencomprises a higher concentration of beta fibrinogen as compared to aconcentration of alpha fibrinogen and a concentration of gammafibrinogen.
 16. The medical device of claim 12, wherein theproteinaceous material comprises albumin.
 17. The medical device ofclaim 12, wherein the proteinaceous material comprises albumin andfibrinogen.
 18. The medical device of claim 12, wherein theproteinaceous fluid is selected from the group consisting of blood,plasma, blood serum, a fluid comprising a blood protein, a fluidcomprising albumin, and a fluid comprising fibrinogen.
 19. The medicaldevice of claim 12, wherein the medical device is selected from thegroup consisting of a heart valve, a vascular stent, a graft, acatheter, and a device comprising a stent.
 20. The medical device ofclaim 12, wherein the at least one anti-thrombogenic property isselected form the group consisting of inhibiting platelet adhesion,inhibiting platelet aggregation, inhibiting thrombosis, inhibitingclotting, and inhibiting coagulation.